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The underlying mechanisms of isoflurane neurotoxicity in the developing brain remain unclear. Ferroptosis is a recently characterized form of programmed cell death distinct from apoptosis or autophagy, characterized by iron-dependent reactive oxygen species (ROS) generation secondary to failure of glutathione-dependent antioxidant defenses. The results of the present study are the first to demonstrate in vitro that ferroptosis is a central mechanism contributing to isoflurane neurotoxicity. We observed in embryonic mouse primary cortical neuronal cultures (day-in-vitro 7) that 6 h of 2% isoflurane exposure was associated with decreased transcription and protein expression of the lipid repair enzyme glutathione peroxidase 4. In parallel, isoflurane exposure resulted in increased ROS generation, disruption in mitochondrial membrane potential, and cell death. These effects were significantly attenuated by pre-treatment with the selective ferroptosis inhibitor ferrostatin-1 (Fer-1). Collectively, these observations provide a novel mechanism for isoflurane-induced injury in the developing brain and suggest that pre-treatment with Fer-1 may be a potential clinical intervention for neuroprotection.

All animal experiments were approved by Stanford University Animal Care and Use Committee (Stanford, CA, USA) and conducted according to the National Institutes of Health guidelines for animal welfare. Primary cortical neuronal cultures were prepared from embryonic/gestational day 15 or 16 Swiss Webster mice as previously described (Stary et al., 2015). Please refer to Supplementary Material for detailed methods.

Total RNA was extracted and reverse transcription and PCR were performed as previously described (Ouyang et al., 2012a,b). Ct-values for GPX4 were normalized to GAPDH as the internal control and comparisons calculated as the inverse log of the ΔΔCT (Livak and Schmittgen, 2001). Please refer to Supplementary Material for detailed methods.

Immunoblot

Please refer to Supplementary Material for detailed methods. After protein gel electrophoresis and transfer membranes were blocked and incubated at 4°C overnight with primary antibodies to GPX4 (1:500, #125066; Abcam) and anti-β-actin (1:20,000, #A1978; Sigma). Membranes were then incubated with 1:3,000 goat anti-rabbit (CST, #7074) for GPX4 and horse anti-mouse (CST, #7076) for β-actin. GPX4 band intensity was normalized to β-actin and the isoflurane group then normalized to the carrier gas group.

Subsequent to isoflurane or carrier gas exposure, cultures were incubated with Hoechst 33342 (5 μM, Sigma) and propidium iodide (PI, 5 μM, Sigma). Automated fluorescent image capture was performed at 200X using a LumascopeTM 720 (Etaluma, Carlsbad, CA). The number of PI-positive and Hoechst-positive cells were quantified using Image J software (v1.49b, National Institutes of Health, USA) and expressed as percentage of total cells.

Assessment of Reactive Oxygen Species (ROS) and Mitochondrial Membrane Potential

All results are expressed as mean ± standard error (SE). Statistical analysis was performed using SPSS 18.0 software. GPX4 mRNA and protein expression levels, CellROS and TMRE fluorescent values were normalized to those of the control group (carrier gas, vehicle alone). All data represent pooled data from 3 individual experiments containing n = 4 samples for each treatment group. After normality and equal variance tests, statistical differences between two groups were compared using Student's t-test. For data with non-normal distributions, Kruskal–Wallis test was used. For all measurements p < 0.05 (95% confidence interval) were considered significant. Please refer to Supplementary Material for detailed methods.

Ferroptosis is defined by iron-dependent accumulation of lipid peroxides, and is genetically and biochemically distinct from other forms of programmed cell death such as apoptosis, necrosis and autophagy (Yang and Stockwell, 2016). GPX4 is a lipid repair enzyme, which reduces lipid hydroperoxides to lipid alcohols and limit the initiation of ferroptosis (Cao and Dixon, 2016; Tonnus and Linkermann, 2016). Inhibition of GPX4 activity leads to rapid accumulation of lipid peroxides and cell death, and deletion of GPX4 is embryonic lethal (Seiler et al., 2008; Yang et al., 2014), therefore GPX4 expression has been used as a marker for ferroptosis (Cardoso et al., 2017; Conrad et al., 2018). In the present study we observed a significant decrease in GPX4 mRNA and GPX4 protein expression with isoflurane exposure (Figure 1), supporting our hypothesis that isoflurane induces ferroptosis. These observations, in combination with our parallel observations that mitochondrial dysfunction secondary to isoflurane exposure were largely reversed by pre-treatment with Fer-1 (Figure 2), imply a central role for ferroptosis in isoflurane neurotoxicity. Future in vivo studies selectively altering GPX4 expression are needed to more accurately assess the mechanistic contribution of decreased GPX4 to isoflurane neurotoxicity.

Whether general anesthetics have the capacity to permanently impair cognitive function remains controversial (Hudson and Hemmings, 2011; Zhou et al., 2015), and clinical trials in matched cohorts testing protective compounds may provide the only avenue for direct evidence of neurotoxicity in children. In the present study we observed that Fer-1 had no effect on ROS generation, mitochondrial membrane potential or cell death in neuronal cultures, suggesting that Fer-1 may provide a clinically safe therapeutic intervention. However, one limitation of the present study is that cell cultures were grown at atmospheric partial pressure of O2 (21%), which represents a hyperoxic state relative to physiological O2 levels in the brain which approach 1–8% (Erecinska and Silver, 2001). Our group have previously described differences in mitochondrial function between cultures grown at 21% and cultures grown at 5% O2 (Sun et al., 2015), suggesting that in vitro experiments assessing mitochondrial mechanisms should account for this. Second, although 2% isoflurane in vitro treatment represents in vivo physiologic levels of exposure (Herold et al., 2017), a 6 h duration of single agent exposure would not represent a typical pediatric anesthetic. Further studies extending these findings to clinically relevant in vivo models utilizing multiple anesthetic agents and more common exposure durations are required to advance Fer-1 as a potential neuroprotectant in pediatric patients requiring anesthesia.